1. Field of the Invention
The present invention relates to a novel sulfotransferase enzyme responsible for sulfation of sugars of glycolipids, the gene encoding said enzyme isolated from a human cell, and production of the sulfotransferase by using an expression vector.
The present invention also relates to methods for control of the expression of sulfotransferase by using an antisense DNA or antisense RNA and methods for detection of sulfotransferase by using a synthetic oligonucleotide probe or primer, as well as an antibody or its fragment.
2. Discussion of the Related Art
In recent years, various physiological functions of the sugar chain moieties of molecules in cell membrane known as complex carbohydrates, such as glycoproteins and glycolipids, have drawn attention. The sulfate group bound to a sugar chain is interesting in terms of various biological functions. The sulfate group is bound to the sugar chains of mucin, mucopolysaccharides and glycolipids in various modes of binding, as well as to the sugar chains of viral glycoproteins, glycoprotein hormones, basement membrane glycoproteins, slime mold lysosome enzymes, etc., via ester linkage. However, its biological significance remains to be elucidated.
Various sulfotransferases of different substrate specificities are already known. For example, sulfotransferases acting on glycoproteins include the sulfotransferase which adds the sulfate group to the 3-position of galactose of N-glycoside type sugar chains [Journal of Biological Chemistry, 264 (6), 3364-3371 (1989)], that which adds the sulfate group to the 6-position of N-acetylglucosamine of N-glycoside type sugar chains [Biochemical Journal, 319, 209-216 (1996)], that which adds the sulfate group to the 3-position of N-acetylgalactosamine of mucin sugar chains [Glycobiology, 5 (7), 689-697 (1995)], that which adds the sulfate group to the 3-position of N-acetylglucosamine of mucin sugar chains [Journal of Biological Chemistry, 270 (46), 27544-27550 (1995)], and that which adds the sulfate group to the 4-position of N-acetylglucosamine of glycoprotein hormone sugar chains produced in the pituitary [Journal of Biological Chemistry, 266 (26), 17142-17150 (1991)].
Sulfotransferases acting on glycosaminoglycans (mucopolysaccharides) include the sulfotransferase which adds the sulfate group to the 2-position of iduronic acid of heparan sulfate [Journal of Biological Chemistry, 271 (13), 7645-7653 (1996)], that which adds the sulfate group to the 6-position of N-sulfated glucosamine of heparan sulfate [Journal of Biological Chemistry, 270 (8), 4172-4179 (1995)], that which adds the sulfate group to the 2-position of iduronic acid of heparin and to the 6-position of N-sulfated glucosamine [Journal of Biological Chemistry, 269 (40), 24538-24541 (1994)], that which adds the sulfate group to the 3-position of N-sulfated glucosamine of heparan sulfate [Journal of Biological Chemistry, 271 (43), 27072-27082 (1996)], that which acts in the N-sulfation of heparan sulfate [Journal of Biological Chemistry, 263 (5), 2417-2422 (1988)], that which acts in the N-sulfation of heparin [Journal of Biological Chemistry, 266 (13), 8044-8049 (1991)], that which adds the sulfate group to the 6-position of N-acetylgalactosamine of chondroitin sulfate and galactose of keratan sulfate and that which adds the sulfate group to the 4-position of N-acetylgalactosamine of chondroitin sulfate [Journal of Biological Chemistry, 268, (29), 21968-21974 (1993)], and that which acts on corneal keratan sulfate only [Journal of Biological Chemistry, 259, (19), 11771-11776 (1984)].
In addition to the sulfotransferase of the present invention, sulfotransferases involved in the synthesis of sugar chains recognized by the monoclonal antibody HNK-1 by adding the sulfate group to the 3-position of glucuronic acid [Journal of Biological Chemistry, 268 (1), 330-336 (1993)] are known to act on glycoplipids.
Of these sulfotransferases, the N-sulfotransferase involved in the synthesis of heparin sugar chains derived from the rat liver [N-heparan sulfate sulfotransferase, Journal of Biological Chemistry, 267 (22), 15744-15750 (1992)], the N-sulfotransferase involved in the synthesis of heparin sugar chains derived from MST cells [N-deacylase/N-sulfotransferase, Journal of Biological Chemistry, 269 (3), 2270-2276 (1994)], and the enzyme involved in the synthesis of chondroitin sugar chains derived from chick embryo chondrocytes by transferring the sulfate group to the C-6 position of N-acetylgalactosamine [chondroitin 6-sulfotransferase, Journal of Biological Chemistry, 270 (31), 18575-18580 (1995)] are known to act on already cloned complex carbohydrates.
The present inventors purified 3'-phosphoadenosine-5'-phosphosulfate:GalCer sulfotransferase [EC 2.8.2.11] a sulfotransferase which adds the sulfate group to the 3-position hydroxyl group of galactose, from a human renal cancer cell line (SMKT-R3) [Journal of Biochemistry, 119 (3), 421-427 (1996)]. Said sulfotransferase is expressed at high levels in human renal cancer tissue or the cell line thereof, which levels are correlated with the accumulation of sulfated glycolipids in renal cancer. Although this fact suggests a relation between the enzyme and cancer, the enzyme's amino acid sequence remains to be determined and the gene therefor remains to be cloned.
In past attempts of industrially advantageous production of known sulfotransferases, it has been very difficult to isolate the desired sulfotransferase in a pure form by simple procedures and in large amounts because of the low natural abundance of the enzyme and the co-presence of other enzymes, such as proteases and sulfatases.
There is therefore a need for a method for cloning sulfotransferase genes and producing sulfotransferases at low cost and high purity using gene engineering technology. Although cloning of a sulfotransferase gene has been reported as stated above, the number of available reports is very few. Also, because there are a large number of sulfotransferases of different substrate specificities, an attempt to use the sequence of one of the above-mentioned sulfotransferase genes to obtain the gene for another sulfotransferase of different substrate specificity encounters a difficulty in obtaining the desired sulfotransferase gene due to the low gene homology between enzymes of different substrate specificities. Moreover, because the amino acid sequences and gene structures of sulfotransferases acting on glycolipid sugar chains remain unknown, such sulfotransferases are difficult to clone and produce by gene engineering.
Accordingly, the first object of the present invention is to provide a gene encoding a sulfotransferase which particularly expresses in renal cells, at higher levels in renal cancer cells and acts on the sugar chain of glycolipids.
The second object of the present invention is to provide a method for producing a sulfotransferase of high purity which is obtained by genetic engineering using a transformant to which an expression vector containing the preceding gene is introduced.
The third object of the present invention is to provide a polypeptide encoded by the preceding gene.
The fourth object of the present invention is to provide an antisense DNA and antisense RNA complementary to the gene of the present invention or a portion thereof.
The fifth object of the present invention is to provide a synthetic oligonucleotide probe or primer specifically hybridizing to the gene of the present invention.
The sixth object of the present invention is to provide an antibody or a fragment thereof which specifically binds to the polypeptide.
The seventh object of the present invention is to provide a purified sulfotransferase.
These and other objects of the present invention will be apparent from the following description.